Many human genetic diseases (about 15%) are caused by genetic mutations that, by interfering with the correct messenger RNA intracellular maturation, compromise the accurate subsequent protein biosynthesis and induce synthesis of non-functional proteins. Mostly, the point mutations accountable for splicing defects concern gene sequences that are critical for the recognition of the primary transcript by the machinery appointed for processing the same. The donor and acceptor sites located at the exon-intron boundaries, as well as gene-specific regulatory elements in exons or introns (Cartegni L et al., 2002; Pagani et al., 2004) are among the most significant sequences. As a consequence of these mutations, various molecular events, which most frequently concern the exclusion of one exon from the mature transcript, the so-called exon skipping, may be induced.
It has been known for a long time that molecular changes in the processing of messenger RNA, which involve for instance exon skipping, represent the main etiopathogenic mechanism of various human diseases, among which hemophilia B, cystic fibrosis, and spinal muscular atrophy, which share the seriousness of their clinical courses. Different types of mutations can induce exon skipping, and specifically mutations in the donor site (or 5′ splicing site), mutations in the acceptor site (3′ splicing site), or exonic mutations. As examples of different types of mutations that induce exon skipping, following are described three models of human diseases.
The defect in the coagulation factor IX (FIX) accounts for the onset of hemophilia B, a disease accompanied by varying degrees of hemorrhagic manifestations, sometimes very serious and disabling. In some cases, the disease is caused by splicing defects. In particular, the exclusion of exon 5 from mRNA during the splicing process is caused both by mutations at position −2 within the exon 5 donor site of the factor IXgene (F9), and by mutations at positions −8 and −9 within the poly-pyrimidine sequence in the acceptor site.
The limitations of the current hemophilia B therapy, which is mainly based on the frequent infusion of recombinant exogenous FIX or of FIX directly derived from plasma, emphasize the need of developing alternative approaches that are characterized by a greater efficacy and a long-lasting effect.
Cystic fibrosis (CF) is the most frequent lethal congenital hereditary disease in the Caucasian population: one newborn out of 2500-2700 born-alive infants is affected by it.
The pathogenesis of this disease is secondary to an anomaly of a protein designated as CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) localized in the apical membrane of epithelium cells and having the function of regulating the hydroelectrolytic exchanges.
As a consequence of CFTR modification, the transfer of salts through cell membranes is compromised, mainly causing a production of secretions that could be defined as “dehydrated”: a sweat very rich in sodium and chlorine and a dense and viscous mucus that tends to obstruct the ducts, compromising the function of various organs and systems. In the course of several studies, many modifications in the CFTR gene sequence were identified as associated with cystic fibrosis, which induce exon skipping. In particular, skipping of exon 12 is caused both by mutations localized within the splicing donor site of the exon itself, and by exonic mutations.
Spinal muscular atrophy (SMA, OMIM 253300, 253550, and 253400) is a recessive autosomal neuromuscular disease characterized by degeneration of spinal marrow alpha moto-neurons, with an estimated prevalence of 1/10,000 born. SMA is associated with clinical syndromes that range from extremely serious, with critical muscle hypotonia and weakness since birth, to milder forms in which the onset occurs later during childhood or adolescence. To date, no treatment for this disease, which generally leads to death at an age that depends on the seriousness of the case history, has yet been identified.
In 95% of cases, the disease is caused by absence of the SMN1 gene. In the human genome, there is a gene homologous to SMN1 called SMN2. However, expression of SMN2 is impaired by a synonymous mutation in the exon which results in an aberrant maturation of the messenger RNA with consequent skipping of exon 7 and inactivation of the gene itself. Approaches designed to increase the number of exon 7-containing SMN2 transcripts would therefore allow to apply a compensation therapy for the absence of the SMN1 gene thanks to the correct expression of SMN2, with considerable implications for a potential effective treatment for SMA.
During the splicing process, the small nuclear RNAs (snRNAs) play a primary role as essential components of the spliceosome, the cell machinery appointed to mediate the entire mRNA maturation process. In particular, the small U1 RNA (U1snRNA), 164 ribonucleotides in length, is encoded by genes that occur in several copies within the human genome and represents the ribonucleic component of the nuclear particle U1snRNP. The U1snRNA molecules have a stem and loop tridimensional structure and within the 5′ region they include a single-stranded sequence, generally 9 nucleotides in length, capable of binding by complementary base pairing the splicing donor site on the pre-mRNA molecule (Horowitz et al., 1994). FIG. 1 shows a schematic representation of the wild-type U1snRNA structure. The sequence in the 5′ region capable of recognizing the splicing donor site is shown paired with the consensus sequence of the splicing donor site in the primary transcripts of eukaryotic genes. Such a sequence exhibits varying degrees of conservation and is located at the exon/intron junction. The recognition mediated by the U1snRNA 5′ region is critical for defining the exon/intron junctions on the primary transcript and for a correct assembly of the spliceosome complex.
The increasing number of human genetic diseases associated with pre-mRNA splicing defects, and the frequent seriousness of the clinical course of the same, stimulated in the last few years the research for therapeutic molecules aimed at correcting splicing defects at the molecular level.
The use of modified U1snRNA molecules capable of inducing in vitro the correct inclusion of the exon and restoring the correct splicing of coagulation factor VII mRNA in case of mutations located at the 5′ss site is described in Pinotti M et al. 2008 and Pinotti M et al., 2009. The illustrated mechanism is based on the recognition and binding of the modified U1snRNA directly onto the 5′ mutated splicing site. However, this method presents a certain degree of non-specificity of action of the therapeutic snRNA molecule towards the target gene, due to the relative conservation of the 5′ ss sites and consequent risk of interfering with the maturation of transcripts generated from other functional wild-type genes. Moreover, it requires the use of a U1snRNA modified for each mutation in the 5′ ss.